Simultaneous concentration and separation of proteins in a nanochannel.

Molecular separation technologies such as gel electrophoresis and liquid chromatography coupled with mass spectrometry detection have been the foundations of biomarker discovery. This is because medically significant biomarkers, for example in blood, can be as much as 10 fold less common than the most abundant protein, albumin and detecting these low abundance molecules requires high sensitivity and selective depletion of the dominant species. Conventional approaches including antibody depletion remove selected molecules by less than 3 orders of magnitude only. This prevents the isolation, characterization and discovery of millions of new proteins where key disease markers could be identified. Overcoming this barrier requires new approaches to analytical detection that minimize sample pre-processing steps while achieving high throughput with very high levels of sensitivity. Here we describe a new device that demonstrates simultaneous concentration and separation of proteins by conductivity gradient focusing without membranes, external pumps, temperature gradients or ampholytes. Concentration and separation take place in an electric field driven, 120-nm deep nanochannel, supporting a stable salt and conductivity gradient. Conductivity gradient focusing is one of many techniques that use opposing convective flow and electrophoretic forces to focus molecules to an equilibrium position. These methods include a step change in chromatographic packings, electrochromatorgraphy, varying the molecular charge (as in isoelectric focusing), temperature gradient focusing, varying the cross section through which the electric current flows, and varying the buffer conductivity. In contrast to all of these approaches, the device presented here does not require ampholytes, matrices or gels, membranes, temperature gradients, or an external pump. Electrokinetic phenomena at the nanoscale have recently been shown to produce rapid and high preconcentration of proteins and peptides in physiological buffers. In these reports nanochannels in microfluidic devices create gradients in the electric field by their charge selective transport characteristics. By combining this with a transport mechanism, often electro-osmosis, charged molecules can be trapped and accumulated owing to a balance in the viscous drag force and the electrophoretic force. The interaction of surface charges, mobile charges, and water molecules with each other and the electric field is complex but our understanding has been advanced by a number of excellent fundamental studies. Concentration polarization, as it is sometimes known, at the entrance to a nanochannel, gives rise to a gradient in the concentration of salt ions, which, in turn, perturbs the electric field creating a trap. Typically these traps cause sample stacking on the microchannel side of the microto nanochannel junction. In such cases the electric field gradient is very abrupt, causing all molecules to accumulate in a tightly confined region with limited scope for separation.